Variation in Growth and Spore Production in the Fungal Pathogen
Alternaria brassicicola

Luke
BarrettCPBR Summer ScholarshipSupervisor: Peter Thrall

Introduction

The study of fungal diseases in natural plant communities can contribute significantly
towards the understanding of evolutionary and ecological dynamics that occur
within plant host-pathogen systems. In this study, I have investigated how variation
in aspects of growth of the fungal pathogen, Alternaria brassicicola may
provide insight into the evolution and ecology of that species. This project
is part of a broader study aiming to address questions regarding the role of
population and spatial structure on disease dynamics within the Cakile maritima-Alternaria
brassicicola host-pathogen interaction on the southern coast of NSW. Because
Cakile only grows on beach foredunes and strandlines, it is easy to delineate
populations, making this system ideal for spatial studies. The study area includes
60 populations in 3 subregions along the south coast of NSW. As part of the
broader study, a large collection of pathogen isolates was generated to examine
spatial structure between populations. These isolates are stored in silica gel
and are easy to re-culture for experimental purposes. While isolates were being
cultured, it was noticed that there was considerable variation in growth rates.
This raised the question of whether isolates which varied in growth rate in
culture also differed in their ability to infect plants, the severity of disease
caused, and in the amount and rate of spore production.

Equally, it is of interest to ask by what means this variation is being maintained
within the broader population. If, for example, higher growth rate leads to
a competitive advantage, with fast growing isolates able to more effectively
colonise and utilise host tissue than slow growing isolates, then directional
selection for faster growing isolates may be expected. Trade-offs between components
of fitness may be one means by which diversity is maintained within natural
populations. Genetic trade-offs could maintain polymorphisms for pathogen aggressiveness
within pathogen populations, since genetic gains associated with one fitness
component, may be offset by genetic losses associated with another fitness component,
resulting in equal fitness overall. For example, while aggressive pathotypes
may be able to more effectively compete for limiting resources, they may be
less efficient at producing spores than less aggressive pathotypes. Selection
may then favour each pathotype equally, but for different reasons. Genetic costs
of increased aggressiveness may be demonstrated in this case through a significant
negative correlation between growth rate in culture and spore production in
culture, providing that a positive correlation can be shown between growth rate
in culture and aggressiveness on the plant.

To explore these possibilities a study was designed to investigate variation
in growth and spore production in the Alternaria isolates. The data collected
were used to address the following questions:

Are the growth rates of A. brassicicola isolatesin culture
indicative of aggressiveness and pathogenicity on Cakile?

Is there a relationship between growth rate in culture, lesion growth rate,
disease severity on the plant and spore production in fungal isolates?

Is there a trade off between growth rate and spore production.

Are there any among population and among region differences in growth dynamics,
and what significance to these have?

Laboratory Trials

The study involved both laboratory and glasshouse components. The laboratory
component involved culturing a range of the available isolates and characterising
them for growth. Alternaria brassicicola isolates were cultured on Potato
Dextrose Agar (PDA). All isolates were grown under identical conditions for
10 days in a 25oC growth chamber. Colony diameters were then measured
along two perpendicular lines. The distribution of growth rates within the entire
set of isolates was determined. Considerable variation in growth rates was observed,
with significant among-population differences.

Because disease epidemic development was censused at regular intervals
in the natural populations from which these isolates were collected, I was able
to compare the mean growth rate of isolates in culture for separate populations,
and the rate of epidemic development within those populations. A significant
negative relationship between epidemic development and mean population growth
rates was evident.

A subset of 20 isolates, ten fast growing and ten slow growing, were then selected
from each end of the distribution in order to establish that fast and slow growing
isolates were significantly different from each other, and for use in assessing
spore production and performance on Cakile plants. Each treatment was
replicated ten times and grown together in a randomised block design under identical
conditions for 10 days in a 25oC growth chamber. There was a highly
significant difference (P < 0.01) between the fast and slow growing groups.

To assess spore production, conidial counts were made for 5 replicates within
each treatment group. Conidial suspensions were prepared from 10 day old cultures
by adding 2.5 ml of 70% ethanol to each plate and rubbing the surface of each
culture with a spreader bar. Conidia were counted with a hemacytometer. A negative
relationship between growth rate and spore production per unit area was found.

Glasshouse
Trials

In order to assess whether or not growth rate in culture is indicative of aggressiveness
on the plant, a glasshouse inoculation trial was performed. Each of the subset
of twenty isolates was seperately inoculated onto a set of 15 Cakile plants
in order to assess isolate aggressiveness. Cakile plants were grown from
randomly selected and mixed seed. Each isolate suspension was adjusted to a
standard concentration using a hemacytometer. Spore suspensions were then atomised
onto each treatment group. Each treatment group was covered with a tent of clear
plastic, preventing cross-contamination and raising humidity levels. The aggressiveness
of each isolate was assessed by scoring each plant in the treatment group for
percent of diseased tissue. The results from this trial have yet to be analysed.

Conclusions

There is a high degree of variability in growth rate among Alternaria brassicicola
isolates. This variability appears to be under genetic control. In addition
to differences between individual isolates, there are also significant differences
between pathogen populations in terms of their mean growth rates. There is a
negative relationship between mean population growth rate and calculated rate
of disease prevalence increase. A negative relationship between spore production
per unit area and growth rate was also found. Viewed together, these two negative
relationships provide good circumstantial evidence for a trade-off between growth
rate and spore production. Slower growing isolates, while perhaps not able to
compete in terms of growth, may be able to produce spores and hence colonise
new plants more effectively than faster growing isolates.